Mechanism Research Of Human Adipose-derived Exosomes Combined With PF-127 Hydrogel Delivering MiR-148a-3p To Promote Angiogenesis In Skin Wounds

Objective: Skin wound healing is a complex physiological process in which vascular regeneration plays an important role.Exosomes(Exos)can regulate the healing process of skin wounds by delivering substances such as proteins,m RNA and micro RNA(mi RNA).Human Adipose-derived Mesenchymal Stem Cell-derived Exosomes(h ADSC-Exos)has higher levels of mi R-148a-3p expression and can promote vascularization.In addition,Pluronic F-127 hydrogel(PF-127 hydrogel)is a new type of biomaterial that has the effect of binding to other active substances and slow-releasing them,which can be used as a carrier for drugs.This study aimed to investigate the role and mechanism of mi R-148a-3p in exosomes in promoting endothelial cell function and skin wound healing and angiogenesis.Methods: 1.Exosomes was isolated from h ADSCs by ultrafiltration,and then identified.Mi R-148a-3p in h ASDSCs was knocked down by transfecting mi R-148a-3p sponge plasmids,and the exosomes with mi R-148a-3p knocked down were obtained.2.Through bioinformatics analysis,the target gene of mi R-148a-3p and signaling pathway were screened.3.The effects of exosomes,PF-127 hydrogels,mi R-148a-3p and target genes on human umbilical vein endothelial cells(HUVECs)were detected by cell function experiments including CCK-8,cell scratch experiment,tube formation experiment,WB and RT-q PCR.4.The targeted inhibition of mi R-148a-3p on its target gene was verified by dual-luciferase reporter assay,and the expression level of target gene after overexpression and knockdown of mi R-148a-3p were detected by WB and RT-q PCR.WB was used to verify whether mi R-148a-3p and the target gene were involved in the signaling pathway.5.The full-thickness skin injury model of mice was established in vivo,and the healing speed was observed after topical application of exosomes,PF-127 hydrogel and exosomes with mi R-148a-3p knocked down.The wound healing effect and angiogenesis were detected by hematoxylin-eosin(H&E)staining,Masson staining,CD31 immunohistochemical staining and WB.Results: 1.The target gene phosphatase and tensin homologue(PTEN)of mi R-148a-3p and its PI3K/AKT signaling pathway were screened by bioinformatics analysis.2.Exosomes was successfully isolated from untreated and mi R-148a-3p knockdown-treated h ADSCs by ultrafiltration.And the decreased mi R-148a-3p levels in exosomes isolated from knockdown-treated h ADSCs was detected by RT-q PCR.3.Through cell function experiments and WB,RT-q PCR,it was found that exosomes can downregulate the expression of the target gene PTEN through mi R-148a-3p,and activate the PI3K/AKT signaling pathway to promote the proliferation,migration and angiogenesis function of HUVECs.PF-127 hydrogel can’t directly promote the function of HUVECs.4.Through in vivo experiments,it was found that exosomes can promote the healing of mouse skin wounds through mi R-148a-3p,and PF-127 hydrogel combined with exosomes treatment has the most significant effect on wound healing,accelerating the wound healing speed and effect,and enhancing the ability of blood vessel regeneration.Conclusion: The results of this study showed that exosomes in h ADSCs could inhibit PTEN expression and activate the PI3K/AKT signaling pathway through its internal mi R-148a-3p delivering,thereby promoting the proliferation,migration and angiogenesis function of HUVECs,promoting skin wound healing and vascular regeneration.PF-127 hydrogel was used in combination with exosomes for wounds,which had better therapeutic effect.